RUNX1 and CBFB are not only important for leukemogenesis but they are also key regulators of normal hematopoiesis. These two genes are required during the earliest steps of hematopoietic stem cell formation and in subsequent stages of several blood lineages. Multiple studies suggest that dysregulation of the normal transcriptional program controlled by RUNX1 and CBFB is likely to be an important mechanism for leukemogenesis. Therefore, better understanding of the RUNX1/CBFB transcriptional program and the roles of RUNX1 and CBFB in normal hematopoiesis will lead to better understanding of the mechanisms for leukemogenesis. We have been pursuing two specific aims in this project in the last fiscal year. In the first specific aim, we have been studying the role of RUNX1 in the formation of hematopoietic stem cells (HSCs) in zebrafish. In particular, we tried to discover how HSCs could form in the absence of RUNX1. Previous studies suggest that RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during embryo development. For example, Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. Surprisingly, we previously discovered that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval bloodless phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish mutant line, which requires verification in independent mutants. In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated three new zebrafish runx1 mutants using the transcription activator-like effector nuclease (TALEN) technology and the CRISPR technology. These mutant lines harbor frameshift or large deletion mutations, resulting in loss of function of runx1 (runx1-/-). These mutants behaved similarly to the previously described runx1W84X/W84X line, so we are now more confident for the existence of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. We have conducted RNA-seq analysis of the kidney of the runx1-/- fish, where hematopoiesis takes place, and identified potential candidate genes that compensate for the loss of runx1 for hematopoiesis. We are also using lineage tracing and single-cell gene expression profiling technologies to identify the cell population responsible for adult hematopoiesis in the runx1 mutants. In the second aim we have been using human induced pluripotent stem cells (iPSCs) to study the function of RUNX1 in human hematopoiesis. We have generated iPSCs from patients with familial platelet disorder, an autosomal dominant disease with reduced platelet numbers and increased susceptibility to leukemia. We have also been working on the culturing conditions for the differentiation of iPSCs to hematopoietic cells, in collaboration with scientists at the National Center for Advancing Translational Sciences. Finally, we have been using genomic technology to determine the genomic integrity of iPSCs. Specifically we have been trying to address two important questions. First, if the iPSCs harbor more mutations than other cultured cells due to reprogramming process; and second, where the mutations coming from. From the same fibroblast populations we generated iPSCs and fibroblast subclones, which are identical to each other in terms of their tissue origin and the way they were derived, except for the treatment of reprogramming factors in the case of the iPSC generation. We then performed NextGen sequencing analysis of the iPSCs and fibroblast subclones to detect mutations. Using this approach we were able to compare the mutation profile of the iPSCs with that of the fibroblast subclones, and provide a definitive answer to the question of if iPSCs have increased mutation burden. Our data reveal that iPSCs have comparable numbers of mutations as their sister fibroblast subclones. Moreover, we demonstrated that >90% of the mutations detected in the iPSCs and the fibroblast subclones were rare, pre-existing, mosaic variants in the parental fibroblast population. Our data therefore strongly demonstrate that iPSC reprogramming is not mutagenic and iPSCs do not contain increased mutation burden. A manuscript reporting these findings has been submitted for publication.